Safe, effective, and inexpensive topical microbicides are urgently needed to curb the global human immunodeficiency virus type-1 (HIV-1) epidemic. Actinohivin (AH) is an actinomycete-derived lectin. This lectin specifically binds to high-mannose clusters uniquely found on the HIV-1 envelope (Env), thereby eliciting nanomolar antiviral activity against multiple HIV strains. Preliminary analyses revealed that AH has a high safety profile in human peripheral blood mononuclear cells (PBMCs) and in the rabbit vaginal irritation assay. Meanwhile, a translational AH-AH fusion protein (recombinant dimer [rd] AH) was suggested to have stronger and broader anti-HIV-1 activity than the original monomer. Given these high potentials, we hypothesize that rAH and/or rdAH (r/rdAH) are excellent HIV-1 microbicide candidates. This project's goal is to reveal the feasibilities of r/rdAH in terms of manufacture, antiviral efficacy, and safety upon use as a vaginal microbicide. In the R21 phase, we will initially focus on developing a highly efficient, scalable production system for r/rdAH that allows for extensive efficacy and safety studies and possible global use. We will utilize recombinant plant virus-based expression systems and various molecular biological approaches for rapid and high-level expression of high-quality r/rdAH. Upon obtaining bulk r/rdAH active pharmaceutical ingredients with high purity standards, we will analyze HIV-1 neutralization effects against selected R5-type viruses in two in vitro HIV neutralization assays based on Env-pseudotyped virus-reporter gene expression and primary isolate- PBMC infection systems. Next, r/rdAH'cytotoxic, mitogenic, and inflammatory potentials will be tested in PBMCs and/or human cervicovaginal (CV) epithelial cell lines to establish the minimal safety profile. Our success criteria in the R21 phase are: (1) establishing the bulk preparation procedure;(2) demonstrating cross- clade antiviral effects to R5 viruses;and (3) demonstrating no apparent in vitro cytotoxicity, mitogenic activity, or inflammatory potential at >100 times above an average anti-HIV IC50, for plant-made r/rdAH. Upon approval of our transition to the R33 phase, we will comprehensively analyze anti-HIV-1 efficacy of r/rdAH for various modes of HIV-1 infection and transmission, using various in vitro assay systems. In addition, we will investigate potential overlap, complementation, synergy, and antagonism of anti-HIV activities between r/rdAH and other inhibitors toward potential microbicide combination strategies. Finally, we will perform extensive evaluations of r/rdAH upon vaginal application in rabbit and mouse models. We will thoroughly evaluate r/rdAH'vaginal toxicity, inflammatory potential, and stability. Upon determining the maximal tolerated dose of r/rdAH, we will examine their potential immunogenicity and toxicity after a long-term exposure. Potential toxicity to the symbiotic vaginal commensal bacteria, the Lactobacillus species, will be examined. In summary, the proposed studies should answer the question of whether r/rdAH is justified for advanced next-stage preclinical studies. . The proposed studies will analyze the feasibilities of the novel HIV-1-binding lectin Actinohivin and its derivative recombinant dimer, as a candidate vaginal HIV-1 microbicide. The proposed studies should generate a comprehensive data set that will reveal their large-scale producibility, anti-HIV-1 efficacy, and broad toxicity profile upon vaginal application, thereby providing criteria of whether Actinohivin and its derivative are justified for further extensive preclinical and clinical studies.